A SPECIFIC ENZYME ASSAY FOR AMINOPEPTIDASE-M IN RAT-BRAIN

A specific enzyme assay for aminopeptidase M (APM) activity on rat brain membranes has been developed through selective use of enzyme inhibitors. Amastatin was the most potent inhibitor (amastatin > actinonin> MDL73347 > bestatin) for purified porcine kidney APM, giving 98% inhibition at a 6muM concentration, while actinonin, yielded only 57% inhibition at this concentration. Puromycin (10 muM) was used to inhibit puromycin-sensitive aminopeptidase activity in the rat brain membrane preparation. Puromycin (10 muM) had only a slight effect on the K(m) of porcine kidney APM, and had negligible effect on APM velocity at the high substrate concentration (2 mM) used in the APM assay. The assay produced a linear accumulation of product for increasing amount of rat brain membranes used, and for increasing incubation time. The K(m) of APM on rat brain membranes for L-Leucine-p-nitroanilide (0.383 mM) was similar to the K(m) of purified porcine kidney APM (0.558 mM). APM-activity, involved in the metabolism of several biologically important neuropeptides in different brain regions, can be specifically measured with this enzyme assay.